In vertebrates, there are over 15 different myosin II isoforms, each of which contains different myosin II heavy chains (MHC IIs). MHC II isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC II isoforms as well as changes in MHC II isoforms during muscle and neural tissue development. This research program has investigated the regulatory mechanisms responsible for the expression of three nonmuscle MHC II (NMHC II) genes, NMHC II-A, NMHC II-B, and NMHC II-C. We have been studying the transcriptional regulation of NMHC II-A and II-C genes as well as tissue-dependent regulation of alternative splicing of NMHC II-B and C genes. During the course of studying the regulatory mechanism for alternative splicing of NMHC II-B, we found that a new RNA binding protein family, the Rbfox family, plays a critical role for neuron-specific alternative splicing of NMHC II-B pre-mRNA. In this report, we focus on the splicing regulators, Rbfox proteins. Rbfox proteins contain a single conserved RNA recognition motif (RRM) in the central region of the molecule and bind specifically to an RNA penta(hexa)nucleotide (U)GCAUG. There are three genes for Rbfox family proteins in mammals, Rbfox1, Rbfox2 and Rbfox3. Rbfox1 is expressed in brain and striated muscles whereas Rbfox2 is expressed in various tissues including brain and muscles. Notably, Rbfox3 expression is restricted to neural tissues. Biochemical analyses of brain cells sorted by Rbfox antibody staining and histological analyses demonstrated that the expression level of the neuron-specific splice variant of NMHC II-B mRNA correlated better with the level of Rbfox-3 expression rather than with that of Rbfox-1 or Rbfox2 expression. These observations suggest that Rbfox3 contributes to neuron-specific splicing of NMHC II-B mRNA, although brain expresses all three Rbfox proteins. Next we extended our research to study the biological function of Rbfox3 using two model systems. First we made use of mouse embryonic carcinoma P19 cells which are capable of differentiating into neuronal cells following retinoic acid treatment. Neuronal differentiation of P19 cells can be monitored by outgrowth of a long axon-like extension which contains an axonal marker, phosphorylated neurofilaments. During neuronal differentiation, expression of Rbfox3 is induced whereas undifferentiated P19 cells do not express Rbfox3. Rbfox1 is barely detected under both undifferentiated and differentiated conditions and the Rbfox2 expression level is unchanged before and after differentiation. The shRNA-mediated knock-down of Rbfox3 results in a decrease in axon-like extensions and an almost complete elimination of phosphorylated neurofilaments. These results indicate that Rbfox3 is required for neuronal differentiation of P19 cells. To understand the molecular mechanism for Rbfox3 function in neuronal differentiation of P19 cells, we carried out Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) to identify transcriptome-wide target RNAs of Rbfox3 in neuronally differentiated P19 cells. Unexpectedly, first we found that Rbfox3 interacts with a wide-range of RNA sequences other than UGCAUG, and that pri-miRNAs are over-represented as Rbfox3 targets. Rbfox3 binds specifically to a subset of pri-miRNAs and functions as either a positive or negative regulator in pri-miRNA processing. Second we used the chicken embryonic spinal cord to study a role for Rbfox3 in neuronal development in vivo. SiRNA-mediated loss of function and rescue experiments showed that Rbfox3-regulated alternative splicing of Numb, which encodes a signaling adapter protein, plays an essential role in differentiation of motor neurons and interneurons during spinal cord development. During characterization of chicken Rbfox3, we also found that the early chicken embryo expresses two Rbfox3 splice isoforms, the full-length Rbfox3 (Rbfox-full) and the 31 amino acid-deleted Rbfox3 (Rbfox3-d31). The latter has a defective RRM and lacks RNA-binding activity. Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin-proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with Rbfox3-full. In cultured cells, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and that its protein level is regulated in an isoform-specific manner in vivo.